Proteolytic enzyme-immobilization techniques for MS-based protein analysis

Protein digestion utilizing proteases (e.g., trypsin, Lys C and other proteolytic enzymes) is one of the key sample-preparation steps in contemporary proteomics, followed by liquid chromatography coupled to mass spectrometry (MS). Tryptic digestion is traditionally performed in aqueous solutions, usually applying the enzyme and the sample in a 50:1 protein-to-protease ratio. Long digestion times (up to 24 h), auto-digestion sub-products and poor enzyme-to-substrate ratio are common issues with liquid-phase protein-digestion processes. The use of enzymes immobilized onto solid supports can minimize these problems by increasing enzyme-to-substrate ratios, significantly speeding up digestion times and reducing autolysis. The other main goal of protease immobilization is to obtain rugged, efficient enzyme reactors.In this article, we review the most important proteolytic enzyme-immobilization techniques with the main emphasis on fabrication of trypsin microreactors and nanoreactors and their utilization in bottom-up proteomics. We also discuss data reportedly obtained using the various immobilization protocols with respect to enzyme activity and MS-sequence coverage.     

Characterizing nonclassicality and entanglement

In Quantum Optics, the widely accepted definition of nonclassicality is based on the P function of Glauber and Sudarshan. When it fails to be interpreted as a classical probability density, the corresponding quantum state is said to be a nonclassical one. Here we present the first reconstruction of a nonclassical P function of a single-photon added thermal state. We also consider the nonclassical properties of general spacegtime dependent correlation functions of radiation fields. For the detection of these correlation functions, a balanced homodyne correlation technique was proposed. It is shown that the measurable correlation functions also allow one to completely characterize bipartite entangled quantum states with a negative partial transposition. Finally, we present a method for identifying general bipartite entanglement for continuous variables.     

Computational explorations of vinylcyclopropane-cyclopentene rearrangements and competing diradical stereoisomerizations

The rearrangements and stereoisomerizations of four systems, vinylcyclopropane, 4-tert-butylvinylcyclopropane, 5-methylvinylcyclopropane, and 2,5-dimethylvinylcyclopropane, as well as a variety of deuterated derivatives and 1- and 2-methyl-, methoxy-, difluoro-, and amino-substituted species, were studied by density functional theory calculations using the B3LYP functional and the 6-31G basis set. Energies were evaluated with CASSCF(4, 4)/6-31G single point calculations. The major product is obtained by the si pathway. Structures on this path are essentially pure diradical in character. Higher energy diradical species and intermediates are responsible for the scrambling of the stereochemistry. The stereoselectivity of the reaction is increased by substituents which increase the relative energy of the species involved in competing stereoselectivities. The computed secondary kinetic isotope effects reproduce the experimental values reported in the literature.     

Vanadium in marine mussels and algae

A method is presented which is sensitive enough for the determination of vanadium (V) in marine organisms such as mussels and algae. It was sufficiently checked by a reference material and it was applied to V determination in blue mussels and brown algae from the German Bight.     

Group contribution analysis of the damping behavior of homopolymers,statistical copolymers,and interpenetrating polymer networks based on acrylic,vinyl, and styrenic mers

The integral of loss modulus versus temperature plots in the vicinity of the glass transition temperature (loss area, LA) was characterized for various homopolymers, statistical copolymers, and interpenetrating polymer networks (IPNs), based on acrylics, vinyls, and styrenic mers. The quantity LA was found to be a molecular characteristic, governed by the structure of the individual mers. The LA of both IPNs and statistical copolymers obeys an additive relation of the component polymers. A group contribution analysis for LA was developed in which each moiety contributes a specific value to LA. A table derived via this group-contribution analysis permits the prediction of LA values. The results show that backbone motions and the moieties attached directly to the backbone contribute the most to the damping peak, and that long side chains act as diluents.     

Determination of heavy metals in sea water and in marine organisms by flameless atomic absorption spectrophotometry

Summary An ultraquick light-beam oscillograph has been used to track the growing and destruction of the atomic cloud in a graphite tube atomizer. In view of the results obtained it was stated that the analytical signal is mainly determined by the stepwise growing of the atomic cloud and its simultaneous destruction by thermal expansion of the inert gas from the tube. Thermodiffusion and recondensation seem to be much slower processes. The dependence of the analytical signal on the settings of the atomizer or the presence of matrix components and on thermal ballast like the L'vov platform is discussed in view of the above thesis. Finally, some proposals are made to overcome the matrix effects by separating the atomization process from the heating and thermal expansion of the surrounding inert gas. For this purpose atomizers with two separate electric current circuits are recommended.

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Bestimmung von Schwermetallen im Meerwasser und in marinen Organismen durch flammenlose AtomabsorptionsspektralphotometrieXV. Matrixeffekte in Graphitrohr-Atomisatoren und Wege zu ihrer Überwindung

Zusammenfassung Ein schneller Lichtpunktschreiber wurde eingesetzt, um das Entstehen und den Abbau der Atomwolke in einem Graphitrohr-Atomisator zu verfolgen. Aufgrund der erhaltenen Ergebnisse wird geschlossen, daß das analytische Signal im wesentlichen durch das allmähliche Wachsen der Atomwolke und deren gleichzeitigen Abbau infolge thermischer Ausdehnung des Inertgases im Rohr bestimmt wird. Thermodiffusion und Rekondensation verlaufen offenbar langsamer. Die Abhängigkeit des analytischen Signals von den Geräteeinstellungen, von der Gegenwart bestimmter Matrixkomponenten und von thermischem Ballast wie der L'vov-Plattform wird im Lichte der o.a. These diskutiert. Schließlich werden einige Wege zur Behebung der Matrixabhängigkeit vorgeschlagen. Sie zielen auf eine möglichst weitgehende Trennung der eigentlichen Atomisierung vom Erhitzen und Sichausdehnen des umgebenen Inertgases. Hierzu werden Atomisatoren mit zwei getrennten elektrischen Heizkreisen vorgeschlagen.

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XIV. Mitt. Fresenius Z Anal Chem (1982) 310:254–256

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Presented at the Colloquium Spurenanalytik, Konstanz, 7.–9.4. 1981     

Factors which affect the glass transition and damping capability of polymers

The shape of the glass transition plays a critical role in the engineering performance of polymers in sound and vibration damping, as well as other applications. The transition may be affected by fillers, plasticizers, blending, IPN formation, etc. A collection of data, both original and literature, is presented which illustrates how the phenomenon works. Emphasis is placed on the role of the area under the linear loss modulus–temperature curve, which may be evaluated in a fashion similar to other spectroscopic techniques. In addition, the loss area can be significantly affected by morphological factors.